Fig. 5. Anti-u-PAR treatment decreases integrin signaling.

A, Gene array analysis of expression changes of mRNAs involved in integrin signaling. Shown is relative fold-difference in the 19 genes which were significantly regulated at least 1.5-fold in anti-u-PAR compared to control IgG treatment. B, The effect of anti-u-PAR treatment on β₃-integrin and FGFR1 was tested in vivo in the SKOV3ip1 and CaOV3 xenograft models and in vitro in SKOV3ip1 and CaOV3 cells grown on plastic or the 3D omental culture. Anti-u-PAR treatment significantly reduced β₃-integrin and FGFR1 mRNA (qRT-PCR) and protein (immunoblot) levels in xenograft ovarian cancer tumors and in ovarian cancer cells cultured on plastic or the 3D culture. Bar graph shows results from three independent experiments (n=5, in vitro, B) and three animals (in vivo, B) +/− standard deviation. C, The effect of anti-u-PAR treatment on α₅-integrin expression was determined in vivo. Anti-u-PAR treatment significantly reduced α₅-integrin (q RT-PCR) and protein (immunoblot) levels in xenograft ovarian cancer tumors. Bar graph shows results from three animals (in vivo, C) +/− standard deviation. D, Colocalization of u-PAR and α₅-integrin on omental ECM in ovarian cancer cells. SKOV3ip1 cells cultured on omental ECM were stained with FITC-labeled uPA and cofocal immunofluorescence for α₅-integrin was performed. Bar graph shows mean percent of uPA-FITC colocalization with α₅-integrin as calculated using the Imaris colocalization application (details in materials and methods). Bar graphs show mean +/− standard deviation (n=5, in vitro) and are representative of 3 independent experiments. A student’s t-test compares the two treatments noted with brackets. * indicates p<0.01.